Method of obtaining a crystalline sodium penicillin



Patented Oct. 18, 1949 METHOD OF OBTAINING A CRYSTALLINE SODIUMPENICILLIN Oskar Wintersteiner, New Brunswick, N. J., and Max Adler, NewYork, N. Y., assignors to E. R. Squibb & Sons, New York, N. Y., acorporation of New York No Drawing. Application June 15, 1944, SerialNo. 540,543

4 Claims. 1

This invention relates to the potent bacteriostatic and/or bactericidalagents formed during the growth processes of various strains of the moldPenicillz'um notatum.

In 1929, Fleming discovered that a strain of a Penicillium mold growingin ordinary nutrient broth formed a powerful antibacterial substance,which he named penicillin (Brit. J. Exptl. Path. 10, 226-36); and thisname was later applied to the product obtained by Florey and hisassociates at the University of Oxford, by the stationary, surface-padgrowth of Penicillium notatum on shallow layers of media, and shown bythem to be a clinically-useful chemotherapeutic agent (Lancet 239, 226,236, 1940; and Lancet 241, 177, 1941).

More recently, it was found advantageous to grow the mold Penicilliumnotatum in submerged culture, i. e., to incubate the mold while it issubmerged in a liquid nutrient medium and the latter is aerated, byagitation or otherwise (Foster and McDaniel application, Serial No.487,140, filed May 15, 1943, now Patent No. 2,448,790, dated September7, 1948); and the product thus obtained (with a medium comprising cornsteep liquor) is at present commonly called sodiumpenicillin (or, often,merely penicillin).

It is now realized that more than one chemical entity has beenidentified by the specific name penicillin. Thus, the crystallineproduct derived, as described hereinafter, from a sodiumpenicillinobtained by growing Penicillium notatum in submerged culture in asynthetic medium in which has been included sodium sulfite (as describedin McCormack application Serial No. 519,207, filed January 2-1, 1944,now Patent No. 2,437,918, dated March 16, 1948) has the empiricalformula C14Hi9O4N2SNa; while the crystalline product heretofore derived(as described in Wintersteiner and MacPhillamy application Serial No.497,719, filed August 6, 1943, which has become abandoned, and thecontinuation-in-part thereof, Serial No. 540,140, filed June 13, 1944,now Patent No. 2,461,949, dated February 15, 1949) from the productobtained by submerged culture with a medium comprising corn steepliquor, has the empirical formula To prevent further confusion,therefore, the former (sodium sulfite) penicillin has been namedthe beenadopted by the chemical research groups Working in this field in thiscountry.

Though the sodium-penicillin F heretofor obtained (e. g., that obtainedas described in the aforementioned McCormack application) is anexceptionally effective chemotherapeutic (antibiotic) agent, thetherapeutic utility thereof is impaired by the impurities present, andits consequent instability on storage.

It is the object of this invention to provide a method of purifyingsodium-penicillin F, especially sodium-penicillin F obtained by growingthe mold in submerged culture in a synthetic medium in which has beenincluded an inorganic, watersoluble sulfite-type compound; and a furtherobject is to provide crystalline sodium-penicillin F, and a method ofobtaining it; and a still further object is to provide highly-active,substantiallypure, acid-penicillin F and derivatives thereof.

In the practice of this invention, high-potency sodium-penicillin F ispurified by dissolving it in an aqueous water-miscible organic solventof medium polarity, such as aqueous acetone, passing the solutionthrough a chromatographic adsorption column, and recoveringsodium-penicillin F from the lower zones of the'column (by elution witha substantially-neutral aqueous buffer solution). From thispartially-purified sodium-penicillin F (or a comparabl sodiumpenicillinF otherwise obtained), crystalline sodium-penicillin F may be obtainedby crystallization from an aqueous lower alkyl ester of a lower fattyacid; and the product may be recrystallized by dissolving it in theminimum volume of an aqueous water-miscible organic solvent ofmedium'polarity, and adding many volumes of a dry lower alkyl ester of alower fatty acid.

The water-miscible organic solvents of medium polarity utilizable in thepractice of this invention comprise, inter alia, the lower-aliphaticketones (e. g., acetone, methyl ethyl ketone, and diethyl ketone) andthe cyclic ethers (e. g., dioxane), the preferred solvent being acetone.The preferred solvent for the crystallization of sodium-penicillin F isaqueous ethyl acetate.

The following examples are illustrative of the invention (the potencyunit referred to being the Florey or Oxford unit used in th UnitedStates of America for standardizing therapeutic preparations ofpenicillin as prescribed by the U. S. Food and Drug Administration) (a)5 g. of the partially-purified sodium-penicillin F obtained as describedin Example 15 of aforementioned McCormack application (possessing apotency of 600 units/mg, a spe been adsorbed, the chromatogram isdeveloped by washing the column with 95% acetone until it has thefollowing appearance; a narrow, dark-- brown band (I) at the top of thecolumn; below this, a, light-brown zone (II)- ex-tend-i-ng downward toabout of the length of the column; below this, a. light-yellow zone(III) extending downward to about the length of the column; and belowthis, a light-yellow band, then a very faintly ye110W zone (IV)extending to almost the bottom of the column, and terminating in anotherlight-yellow band. The washing with acetone is discontinued after thelatter band has passed into the filtrate.

The acetone washings contain very little activity and are discarded. Thecolumn is sucked dry with an air current, and the various sectionsindicated above are separated. Band I contains very little activity andis discarded. Zone II is eluted with a pH 7 phosphate buffer solution,yielding material of comparatively low potency. Zones III and IV arecombined and eluted with a pH '7 phosphate buffer solution; and thepenicillin F contained in this solution is recovered as the sodium saltby acidifying the ice-cooled buffer solution with 10% phosphoric acid topH 2-3, extracting three times with ether, washing the combined etherextracts with water, re-extracting with small portions of 1% aqueoussodium bicarbonate solution until the pH of the last extract is 6.5,freezing the combined aqeous extracts, and subjecting, the frozenmaterial to a high vacuum to sublime out the water. The thus-purifiedsodium-penicillin F weighs 1.5 g., has a potency of 1030 units/mg, andhas a specific rotation [(11 of +258". (Similar treatment of the zone IIeluate yields 0.6 g. of a salt having a potency of 83 units/mg, whichmay be combined with the same fraction from other batches andrechromatographed.)

(b) 226 mg. of the sodium-penicillin F obtained as decsribed in section(a)or a sodiumpenicillin F of comparable purity otherwise 0btainedisdissolved in 2.5 cc. dry ethyl acetate, and the very small amount ofdark flocculent matter remaining undissolved is removed by centrifuging.A very small quantity of water, just suflicient to cause a faintturbidity, is added to the supernatant, the solution is allowed to standfor an hour at room temperature (the turbidity increasing slightly), andthen chilled in the refrigerator. In the course of two hours, white,needle-like crystals begin to form; and after standing 12-16 hours inthe refrigerator, the crystalline material is filtered off, washedseveral times with dry ethyl acetate, and dried over calcium chloride invacuo. (The yield of crystalline product is 70 mg.; and an additionalyield of 41 mg. is obtained from the combined filtrate and washingsafter it has been in the refrigerator for several days.)

The product, crystalline sodium-penicillin F, is hygroscopic when freelyexposed to the atmosphere (losing about 1% of its weight on drying at100 in vacuo for 2 hours), and melts at 204-205 C. with decomposition ina capillary tube inserted into the melting-point block at 160 C. It hasthe empirical formula Ci4H19O4N2SNa (a product,

dried in vacuo at C. for 2 hours, giving the following figures onanalysis: C, 50.27; H, 5.67; N, 8.43; S, 9.39; and Na, 6.95; while thecalculated analysis for CiiHiaOiNzsNa is: C, 50.27; H, 5.73; N, 8.38; S,9.59; and Na, 6.88), the specific rotation [(11 of +290 in water, and apotency of 1350' units/mg. The ultraviolet absorption curve of theproduct shows merely end absorption beginning at 2500 A. and reaching anE value of 3000 at 2250 A. (the low maxima at 2630, 2570, and 2520 A.evident in the spectrum of crystalline sodium penicillin G beingabsent). On degradation of the product by hydrolysis with boiling 0.1normal sulfuric acid and subsequent treatment with mercuric chloride andfiltration, an aldehyde of the formula C8Hl302N is obtained in thefiltrate, and may be isolated in the form of its2,4-dinitro-phenylhydrazone of the formula C14H17O5N5 melting at 179 C.

Crystalline sodium-penicillin F, dried in vacuo at 100 C. for 2 hours,is fully as active as the undried material, while impure preparations ofsodium-penicillin F lose most of their activity under these conditions.

The crystalline sodium-penicillin F obtained as described hereinbeforemay be recrystallized by dissolving it in the minimum volume of 98%acetone required for solution, and adding 5 volumes of dry ethylacetate. On recrystallization thus three times, the product has thespecific rotation [011 of +316.5 in water and a potency of 1425'units/me.

The structural formula of sodium-penicillin F S CH3 H NOH-C O'ONa Oalternatively, the purified sodium-penicillin F obtained as described insection (a) may be crystallized by dissolving it in a volume ofwater-sat.- urated ethyl acetate just sufficient for solution, andchilling and further treating the solution as described hereinbefore.

The crystalline sodium-penicillin F obtained in accordance with thisinvention may be used advantageously for the preparation ofhighly-active. substantially-pure acid-penicillin F and deriva- :tivesthereof. Thus, such acid-penicillin F, having the empirical formulaC14H20O4N2S, may be obtained by acidifying an aqueous solution of thecrystalline sodium-penicillin F with a 10% solution of phosphoric acid,extracting the aqueous solution with ether, drying the ether extract,and removing the solvent in vacuo. From this acidpenicillin F, Varioushighly-active, substantiallypure derivatives may be obtained, interalia, esters and other salts. For example, a calcium salt may beobtained by dissolving the acid-penicillin F in ether, extracting theether with small portions of aqueous Ca(OI-I)z or with an aqueoussuspension of CaCOs, and removing the water in vacuo; and the esters maybe obtained by treating a dry ether solution of the acid-penicillin Fwith a diazoalkane, e. g., diazomethane. In the foregoing procedures,all treatments involving acid solutions (acid-penicillin F) are, ofcourse, carried out at low temperature, -e. g., while cooling with ice.

The invention may be variously otherwise embodied within the scope Ofthe appended claims.

We claim:

1'. In the method of obtaining a crystalline sodium-penicillin of thetype characterized by the structural formula o the step of crystallizingan amorphous highpotency sodiumpenicillin of such type from an aqueouslower alkyl ester of a lower fatty acid.

2. In the method of obtaining a crystalline sodium-penicillin of thetype characterized by the structural formula l i (P011.

---11-coo1 m the step of crystallizing an amorphous highpotencysodium-penicillin of such type from aqueous ethyl acetate.

3. In the method of obtaining a crystalline sodium-penicillin of thetype characterized by the structural formula quantity of a substantiallydry lower alkyl ester of a lower fatty acid to cause crystallization.

4. In the method of obtaining a crystalline sodium-penicillin of thetype characterized by the structural formula l CH I N 11-0 0 0N8 thesteps of crystallizing an amorphous high-potency sodium-penicillin ofsuch type from an aqueous lower alkyl ester of a lower fatty acid.dissolving the crystalline product in a minimum volume of aqueousacetone, and adding a suflicient quantity of substantially dry ethylacetate to cause crystallization.

OSKAR. WINTERSTEDIER.

MAX ADLER.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 2,399,840 Wachtel May 7, 19462,437,918 McCormack Mar. 16, 1948 OTHER REFERENCES Lancet H, pp.177-188, Aug. 16, 1941. Abraham: British Journal of ExperimentalPathology, vol. 23, June 1942, pp. 103-125.

Science, July 3, 1942, pp. 20 and 21.

Manufacturing Chemist and Manufacturing Perfumer, Aug. 1943, XIV, pp.251-254.

J. Am. Med. Assoc., p. 736, July 7, 1945.

Separation and Identification of Penicillin Species in CommercialPenicillins (Q. 176) Culture, by Salivar (Feb. 1, 1947).

